Random peptide libraries and protein diversity generated by means of phage display methodology are becoming invaluable for the identification of new small molecule drugs. These approaches that produce a large multiplicity of peptides are encompassed by the term "combinatorial chemistry." Early combinatorial chemistry efforts involved one or more variations on the Merrifield peptide synthesis scheme (R. Merrifield, J. Am. Chem. Soc. 85:2149-54, 1963). This scheme features incremental lengthening of a peptide chain on a solid phase support. Today, commercially available equipment can be used to perform peptide synthesis on solid supports. More specifically, after a first amino acid is attached to a solid support, a series of reactions involving deprotection, attachment of the next amino acid (protected) in the sequence, deprotection of the peptide, and so on, is carried out to generate a synthetic peptide or polypeptide. Peptide combinatorial libraries have been recently reviewed by M. Gallop et al., J. Med. Chem. 37: 1233-51, 1994 and E. Gordon et al., J. Med. Chem. 37: 1386-1401, 1994, for instance.
Large combinatorial libraries (containing millions and even billions of peptides) can now be generated and analyzed. By using standard, solid phase peptide synthesis methods in conjunction with a resin proportioning (split) and mix technique protocol (see, for instance, A. Furuka et al., Abstr. 14th International Congress of Biochemistry, Prague, Czechoslovakia, Vol. 5, p. 47, 1988; A. Furuka et al., Intl. J. Pept. Protein Res. 37:487-93, 1991; K. Lam et al., Nature 354: 82-84, 1991; and R. Houghten et al., Nature 354:84-86, 1991), equimolar mixtures of peptides with one unique sequence per bead can be produced. Deconvolution of these peptide libraries is accomplished by subjecting peptide mixtures (in solution) to bioassays in an iterative fashion (for instance, as described by R. Houghten et al., BioTechniques 13:412-21, 1992). By screening progressively smaller libraries and sub libraries of peptide mixtures, an active sequence(s) can be identified. Alternatively, solid phase-immobilized peptides can be screened using appropriately developed bioassays. Deconvoluted beads displaying a bioactive peptide sequence can then be sequenced using a peptide sequencer (see K. Lam et al., Bioorg. Med. Chem. Lett. 3:419-24, 1993).
There are advantages and disadvantages to each of these processes (i.e., using compounds in solution versus immobilized compounds for bioassay and deconvolution). Compounds in solution offer the following advantages: (1) compounds can be assayed in conjunction with soluble or insoluble receptors, enzymes or cell-based bioassays; (2) the molarity of compounds can be controlled; and (3) deconvolution strategies are readily available. The disadvantages of compounds in solution include: (1) complex mixtures in solution can result in multiple "hits"; (2) many compounds having low activity may be present in the same pool; (3) deconvolution can be elaborate and time-consuming; and (4) total pool concentrations cannot exceed a certain, limiting concentration.
Immobilized or unsolubilized (for instance, on beads) compounds provide the following advantages: (1) ease of detection and selection of positive compounds; (2) no cleavage work-up is needed; (3) each bead or matrix unit is associated with a single compound that can be readily identified. The disadvantages of immobilized compounds include: (1) uncertain effects of the solid support on the activity of affixed compounds; (2) potential conformation constraints on the affixed compound and its activity; (3) the compounds are generally small, and naturally would interact with receptors or binding moieties in a small form, not as a small portion of a larger molecular entity; (4) the detection molecule (for instance, a receptor or enzyme in a bioassay format) must be soluble, and may need to be modified, configured or adapted to function appropriately in a solid phase assay format; and (5) membrane-bound detection molecules (such as receptors or in cell-based assays) may not be amenable to a solid phase assay format.
Methods have been developed for synthesis and deconvolution of nucleotide-encoded peptide libraries (N. Needels et al., Proc. Natl. Acad. Sci. U.S.A. 90:10700-04, 1993; R. Lerner et al., PCT Application WO 93/20242); chemically-tagged peptide libraries (A. Borchardt et al., J. Am. Chem. Soc. 116:373-74, 1994); and peptide-tagged non-sequencible libraries (V. Nikolaiev et al., Pept. Res. 6:161-70, 1993). Also, a multiple use library has been used to screen a G-coupled receptor system (C. Jayawickreme et al., Proc. Natl. Acad. Sci. U.S.A. 91:1614-18, 1994). Many of these libraries and assays have undergone limited testing and analysis. Thus, to make these libraries generally useful in conjunction with a spectrum of bioassays, especially cell-based and/or membrane-bound receptor assays, considerable improvements in these methods are needed.
To overcome limitations associated with reported peptide libraries, there is a need in the art for alternatives to known, peptide-based combinatorial libraries. The present invention provides such alternatives, as well as other, related advantages.